1. Field of the Invention
The present invention relates generally to the fields of molecular biology, oncology and pharmacology. More particularly, it concerns methods for modulating macropinocytosis by modulating the binding of dynein light chain-1/protein inhibitor of nitric oxide synthase (DLC1/PIN) to p21-activated protein kinase 1 (Pak1). It also concerns methods of screening for modulators of macropinocytosis by assessing the ability of candidate substances to modulate the binding of DLC1/PIN to Pak1. In addition, it concerns methods of treatment of cancer, viral infections, and other conditions associated with macropinocytosis wherein targeting DLC1/PIN and Pak1 interaction may be beneficial.
2. Description of Related Art
The p21-activated kinases (Paks), an evolutionarily conserved family of serine/threonine kinases, are important for a variety of cellular functions including cell morphogenesis, cell motility, cell survival, angiogenesis and mitosis (Kumar and Vadlamudi, 2002; Jaffer and Chernoff, 2002). At present, the Pak family consists of six members, Pak 1 through Pak 6. Paks are identified as one of the targets of the activated Rho GTPases Cdc42 and Rac1, which stimulate Pak autophosphorylation and activity (Manser et al., 1994). Stimulation of Pak activity results in several phenotypic changes reminiscent of those produced by Cdc42 and Rac1 (Sells et al., 1997). Overexpression of Paks in cancer cells increases cell migration potential and anchorage-independent growth and causes abnormalities in mitosis (Banerjee et al., 2002; Vadlamudi et al., 2000a; Li et al., 2002a; Thiel et al., 2002).
Paks are widely expressed in numerous tissues and are activated by a number of polypeptide factors and extracellular signals in both a GTPase-dependent (via Rac1 or Cdc42) and GTPase-independent manner via its localization to membrane/focal adhesion (Bokoch et al., 1996; Bagrodia and Cerione, 1999; Zhao et al., 2000). They are also activated by lipids (Bokoch et al., 1998), tyrosine kinases (McManus et al., 2000; Bagheri et al., 2001), novel substrates such as filamin (Vadlamudi et al., 2002b) and G-proteins (Lian et al., 2001). The activation of Pak1 by diverse signals leads to its autophosphorylation at multiple sites, including threonine 423 (T423), within the activation loop of the kinase (Daniels and Bokoch, 1999). Expression of an activated Pak1 mutant (T423E) triggers the dissolution of stress fibers and focal adhesion complexes, the formation of lamellipodia (Manser et al., 1997; Zhao et al., 1998), and reorganization of actin cytoskeleton. Some of the effects of Pak on the actin cytoskeleton appear to be independent of Pak1 kinase activity but dependent on protein-protein interactions (Turner et al., 1999; Sells et al., 1997). However, kinase activity is important for directional motility (Sells et al., 2000). Pak1 mutants with defective GTPase binding sites or kinase activity nonetheless still maintain the ability to induce the formation of membrane ruffles and filopodia (Frost et al., 1998; Sells et al., 1997). Pak1 also stimulates LIM kinase (LIMK) activity and, in turn, increases phosphorylation and inactivation of cofilin, leading to reducing the depolymerization of actin filaments (Edwards and Gill, 1999; Edwards et al., 1999, Jaffer and Chernoff, 2002; Bagheri et al., 2002). However, the molecular mechanism by which Pak1 induces rearrangement of the actin cytoskeleton and directional movement remains elusive.
Reorganization of the cytoskeleton not only affects cell motility but also plays an important role in pinocytosis, a process by which macromolecules and fluids are taken up into small invaginations in the cell membrane that eventually bud off into pinosomes (Swanson and Watts, 2002). Pinocytosis contributes to both the growth and motility processes of cells (Davies et al., 1980; Thompson and Bretscher, 2002). Recently, Pak1 was shown to localize to the areas of pinocytic vesicles and to contribute to the process of macropinocytosis (Dharmawardhane et al., 1997; West et al., 2000). Pak activity was also required for growth factor-induced macropinocytosis, and accordingly, catalytically activated Pak1 enhanced both the uptake and efflux of a 70-kDa dextran particle, suggesting that Pak1 activity modulates pinocytic vesicle cycling (Dharmawardhane et al., 2000). Furthermore, transient stimulation of macropinocytosis by growth factors has been also implicated in directed cell motility, as regulation of membrane flux via Pinocytosis could contribute to the membrane flow generating force for cell locomotion (Thompson and Bretscher, 2002; Bretscher and Aguado, 1998; Dharmawardhane et al., 2000). Although Pak1 has been shown to regulate macropinocytosis, the nature of the responsive molecular mechanism remains unknown.
Cytoskeleton remodeling-dependent cellular processes, such as vesicle transport and membrane transport are also influenced by dynein, a multi protein complex originally shown to regulate the movement of chromosomes, assembly and orientation of mitotic spindles and nuclear migration (Holzbaur and Vallee, 1994; Hayden, 1988; Steuer et al., 1990; Vaisberg et al., 1993; Beckwith et al., 1998). Dynein light chain-1 (DLC1/PIN), an 8-kDa component of the cytoplasmic dynein complex, is a minus end-directed microtubule-based motor that transports cargo along microtubules (Hirokawa, 1998). DLC1/PIN is highly conserved among species and widely expressed in a number of tissues; it is localized predominantly in the cytoplasm. In addition to playing an essential role in dynein motor function, DLC1/PIN interacts with a number of proteins and has diverse functions. For example, DLC1/PIN associates with neuronal nitric oxide synthase (nNOS) and inhibits its activity and proapoptotic function (Jaffrey and Snyder, 1996). DLC1/PIN also interacts and interferes with the proapototic Bcl-2 family protein Bim (Puthalakath et al., 1999). Although the functional role of DLC1/PIN in vesicle trafficking and cell survival has been observed, no function of DLC1/PIN has been associated with its phosphorylation. In addition, no upstream signaling kinase have been shown to phosphorylate DLC1/PIN and influence these functional outcomes.
Macropinocytosis, a cytoskeleton remodeling-dependent cellular process, plays a central role in cellular uptake of nutrients and macromolecules, and membrane flux (Haigler et al., 1979). In addition, regulated cycling of plasma membrane via macropinocytosis has been proposed to have a role in directed cell movement (Bretscher and Aguado, 1998; Dharmawardhane et al., 2000; Thompson and Bretscher, 2002). Macropinocytosis is considered to generally be a nonspecific mechanism for internalization of extracellular material by a cell, in that it is not reliant on ligand binding to a specific receptor (reviewed in Sieczkarski and Whittaker, 2002). Instead, formation of endocytic vesicles occurs as a cell type-specific response to cell stimulation, resulting in the closure of lamellipodia at the sites of membrane ruffling to form the large (0.2 to 3 μm), irregular vesicles known as macroPinosomes (Lanzavecchia, 1996). Membrane ruffling is primarily actin-driven and macropinocytosis is, in terms of mechanics, similar to the process of phagocytosis that occurs in specialized immune system cells such as neutrophils and macrophages.
Macropinocytosis also plays an important role in the uptake of macromolecules in epithelial cells, neutrophils and macrophages (Aderem and Underhill, 1999), in taking up extracellular antigens into antigen-presenting dendritic cells (West et al., 2000), and the entering of human immunodeficiency Type 1 virus (HIV) in macrophages (Marechal et al., 2001) and endothelial cells (Hirokawa, 1998).
The process of macropinocytosis requires the small GTPases and p21-activated kinase 1 (Pak1) (West, 2000; Dharmawardhane et al., 2000). Pak1, an effector of Cdc42/Rac1, specifically regulates macropinocytosis, but not clathrin- or receptor-mediated endocytosis (Dharmawardhane et al., 1997). Even though the mechanism by which Pak1 regulates macropinocytosis is not known the results of earlier studies suggest that Pak1 activity plays an essential role (Dharmawardhane et al., 2000).
Furthermore, intracellular vesicle transport is also regulated by microtubule-based DLC1/PIN, a component of dynein complex originally shown to regulate the movement of chromosomes and orientation of spindles (Hirokawa, 1998; Vallee and Sheetz, 1996; Pazour et al., 1998). DLC1/PIN has also been shown to promote cell survival (Vadlamudi et al., 2000; Vadlamudi et al., 2002). However, no function of DLC1/PIN has been associated with its phosphorylation by upstream signaling kinase and whether such changes might control macropinocytosis in mammalian cells.
The inventors have identified DLC1/PIN as a Pak1-interacting protein and have shown that DLC1/PIN is a physiological interacting substrate of Pak1. The Pak1-DLC1/PIN interactions play an essential role for the macropinocytosis and cell-survival functions of Pak1 and also DLC1/PIN. The inventors have found that the underlying mechanism involves the phosphorylation of serine 88 of DLC1/PIN by Pak1 because mutation of this serine residue to alanine abolishes the ability of DLC1/PIN to support macropinocytosis and cell survival. Unexpectedly, the inventors have found that DLC1/PIN expression is elevated in human breast tumors, and deregulation of DLC1/PIN but not DLC1/PIN mutant lacking serine 88 promotes the tumorigenic potential of breast cancer cells.
Thus, the regulation of macropinocytosis, cell survival and tumorigenic functions by interaction between DLC1/PIN and Pak1 represents a novel mechanism by which a signaling kinase might control these essential processes in cells.